Mechanistic dissection of novel regulators of TDP-43 aggregation identified in a genome-wide CRISPR-Cas9 knockout screen

全基因组 CRISPR-Cas9 敲除筛选中发现的 TDP-43 聚集新型调节因子的机制剖析

基本信息

批准号：
10268168
负责人：
Katelyn Sweeney
金额：
$ 3.35万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-06-01 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10268168
关键词：
ALS patients Address Affect Alzheimer&apos s Disease Amyotrophic Lateral Sclerosis Antiviral Agents Astrocytes Binding Biology CRISPR/Cas technology Cell Line Cells Cellular Morphology Client Coculture Techniques Collaborations Coupled Cyclic AMP-Dependent Protein Kinases Data Development Diffuse Disease Disease Progression Dissection Double-Stranded RNA Enzyme-Linked Immunosorbent Assay Fluorescence-Activated Cell Sorting Generations Genes Genetic Predisposition to Disease Global Change Goals Homeostasis Immune Immune response Immune signaling Importins In Vitro Incidence Interferon-alpha Knock-out Link Mass Spectrum Analysis Measures Mediating Messenger RNA Methyltransferase Microglia Mind Modeling Modification Molecular Chaperones Motor Neurons Mutation Neurodegenerative Disorders Nuclear Nuclear Export Pathogenesis Pathologic Pathology Pathway interactions Phase Physiologic pulse Prevalence Proteins Quality Control RNA RNA Binding RNA methylation RNA-Binding Proteins Regulation Reporter Ribosomal RNA Role Signal Pathway Signal Transduction Small RNA Stains Structure Testing Therapeutic Time Toxic effect Translations Western Blotting Work Yeast Model System Yeasts base crosslinking and immunoprecipitation sequencing disease-causing mutation follow-up frontotemporal degeneration genome-wide in vitro testing interest novel prevent protein TDP-43 protein kinase R shape analysis therapeutically effective

项目摘要

Project Summary The mislocalization and aggregation of RNA-binding proteins (RBP) is a pathological hallmark of Amyotrophic Lateral Sclerosis (ALS). The RBP TAR DNA-binding protein 43 (TDP-43) is of particular interest, as pathological aggregation of TDP-43 is observed in nearly 97% of ALS patients, despite the fact that disease-causing mutations in TDP-43 explain fewer than 5% of disease incidence. This observation suggests that misregulation or mutation of other genes converge on TDP-43 pathology, however it remains poorly understood how the aggregates arise or whether they can be reversed or prevented. To this end, we have developed Pulse-Shape Analysis-based aggregation reporters for the TDP-43 that, when coupled to fluorescence activated cell sorting, quantifies aggregation at the single cell level. This reporter was leveraged against a genome-wide CRISPR- Cas9 knockout screen to identify regulators of TDP-43 aggregation. Reassuringly, this screen revealed known interactors and pathways involved in TDP-43 regulation and pathology. Additionally, this work revealed several novel proteins not previously implicated in TDP-43 biology or aggregation. Initial follow up on one top hit, SRRD, revealed that this protein reduces TDP-43 aggregation in a mammalian aggregation model, and reduces TDP- 43-associated toxicity in a yeast model, indicating that these genes indeed modify TDP-43 aggregation. The goal of this proposal is to mechanistically dissect how these novel regulators modify TDP-43 aggregation. Using an induced motor neuron model of TDP-43 aggregation, yeast toxicity models, and in vitro studies, I will assess the interactors, signaling pathways, and mode of TDP-43 modification for the following four genes: METTL5, EIF2AK2, XPO4, and SRRD. This work will not only reveal the function of uncharacterized proteins, but dissect their heretofore unknown mechanisms in regulating TDP-43 aggregation. Through better understanding of how TDP-43 aggregation is regulated, the field can begin to answer questions regarding the role of TDP-43 aggregation in disease pathogenesis and progression. By understanding the regulatory network of underlying TDP-43 aggregation, we can begin to therapeutically modulate the pathways that contribute to disease.

项目摘要 RNA结合蛋白（RBP）的错误定位和聚集是肌萎缩症的病理标志侧硬化症（ALS）。 RBP TAR DNA结合蛋白43（TDP-43）特别感兴趣，如病理尽管近97％的ALS患者观察到TDP-43的聚集 TDP-43中的突变解释了不到5％的疾病发病率。这个观察表明不调节或其他基因的突变在TDP-43病理学上汇聚，但是它仍然不太了解汇总出现或是否可以逆转或预防。为此，我们开发了脉冲形状 TDP-43的基于分析的聚合报告基因，当偶联与荧光激活的细胞分类耦合时量化单细胞水平的聚集。这位记者对全基因组CRISPR的利用。 CAS9敲除屏幕以识别TDP-43聚合的调节剂。令人放心的是，此屏幕揭示了已知 TDP-43调节和病理学涉及的相互作用和途径。此外，这项工作揭示了几个以前不与TDP-43生物学或聚集有关的新型蛋白质。最初的跟进一击，SRD，揭示该蛋白质会在哺乳动物聚集模型中降低TDP-43的聚集，并降低TDP- 酵母模型中43个相关的毒性，表明这些基因确实改变了TDP-43聚集。该建议的目的是机械地剖析这些新型调节器如何修改TDP-43 聚合。使用TDP-43聚集的诱导运动神经元模型，酵母毒性模型和体外研究，我将评估以下四个的交互作用者，信号通路和TDP-43修改模式基因：mettl5，eif2ak2，xpo4和srrd。这项工作不仅会揭示未表征的功能蛋白质，但在调节TDP-43聚集时剖析其迄今未知机制。通过更好了解如何调节TDP-43聚合，该领域可以开始回答有关该领域的问题 TDP-43聚集在疾病发病机理和进展中的作用。通过了解基础TDP-43聚合的监管网络，我们可以开始治疗调节导致疾病的途径。